PROJECT SUMMARY RNA binding proteins (RBPs) bind to non-coding, pre-, and mature RNA within the cell to regulate each step of RNA processing, including pre-mRNA alternative splicing, RNA stability and localization, and control of translation. It has become clear that altered RNA processing plays critical roles in nearly every studied biological system, and recent work has suggest that a substantial fraction of disease-causing genetic mutations affect RNA processing, including mutations that cause familial Spinal Muscular Atrophy, Amyotrophic Lateral Sclerosis, and multiple cancer types. Mechanistic understanding of the downstream regulatory network of an RBP is essential to studying and, ultimately, ameliorating these diseases; however, there remains a need for robust, unbiased genome-wide methods to characterize RBP targets and regulators. Building upon our recent development of enhanced crosslinking and immunoprecipitation (eCLIP), I propose to extend this work in three unique directions that each contribute to our ability to gain global, high-quality views of RNA processing transcriptome-wide: 1. Develop low-sample and tag-eCLIP methods for highly parallelizable in vivo profiling of RBPs in low input samples, and for RBPs which lack high-quality native antibodies for immunoprecipitation. 2. Show that transcriptome profiling coupled with RBP target identification can identify critical regulators of a biological system, using differentiation of human induced pluripotent stem cells as a model system 3. Develop methods for unbiased identification of upstream functional regulators of non-coding RNAs and RNA processing in an RNA-centric manner. My extensive expertise in genomics, computational biology, and the study of DNA and RNA binding proteins makes me an ideal candidate to perform the research proposed above. These three aims take different approaches that will coalesce in a robust ability to begin either with an RBP of interest and identify its regulated targets, or begin with an RNA of interest and identify regulator RBPs, which will serve as the basis for my independent research program as an independent faculty candidate. The Yeo lab at UCSD is an ideal environment to perform this research and complete my training towards pursuit of an independent academic faculty position, as it has consistently been a leader in developing both experimental and computational methods to characterize RBP regulation. Additionally, the location of the Yeo lab proximal to outstanding researchers at UCSD, the Salk Institute, and other research institutes and biotechnology companies in La Jolla will provide specific hands-on experimental training in stem cell culture and differentiation, as well as ample opportunities for mentored training in performing research and developing an independent research program.